Journal of Cerebral Blood Flow & Metabolism

Traumatic brain injury (TBI) is an expanding public health epidemic with pathophysiology that is difficult to diagnose and thus treat. TBI biomarkers should assess patients across severities and reveal pathophysiology, but currently, their kinetics and specificity are unclear. No single ideal TBI biomarker exists. We identified new candidates from a TBI CSF proteome by selecting trauma-released, astrocyte-enriched proteins including aldolase C (ALDOC), its 38kD breakdown product (BDP), brain lipid binding protein (BLBP), astrocytic phosphoprotein (PEA15), glutamine synthetase (GS) and new 18-25kD-GFAP-BDPs. Their levels increased over four orders of magnitude in severe TBI CSF. First post-injury week, ALDOC levels were markedly high and stable. Short-lived BLBP and PEA15 related to injury progression. ALDOC, BLBP and PEA15 appeared hyper-acutely and were similarly robust in severe and mild TBI blood; 25kD-GFAP-BDP appeared overnight after TBI and was rarely present after mild TBI. Using a human culture trauma model, we investigated biomarker kinetics. Wounded (mechanoporated) astrocytes released ALDOC, BLBP and PEA15 acutely. Delayed cell death corresponded with GFAP release and proteolysis into small GFAP-BDPs. Associating biomarkers with cellular injury stages produced astroglial injury-defined (AID) biomarkers that facilitate TBI assessment, as neurological deficits are rooted not only in death of CNS cells, but also in their functional compromise.

The vascular basement membrane contributes to the integrity of the blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs). The BCECs receive support from pericytes embedded in the vascular basement membrane and from astrocyte endfeet. The vascular basement membrane forms a three-dimensional protein network predominantly composed of laminin, collagen IV, nidogen, and heparan sulfate proteoglycans that mutually support interactions between BCECs, pericytes, and astrocytes. Major changes in the molecular composition of the vascular basement membrane are observed in acute and chronic neuropathological settings. In the present review, we cover the significance of the vascular basement membrane in the healthy and pathological brain. In stroke, loss of BBB integrity is accompanied by upregulation of proteolytic enzymes and degradation of vascular basement membrane proteins. There is yet no causal relationship between expression or activity of matrix proteases and the degradation of vascular matrix proteins in vivo. In Alzheimer’s disease, changes in the vascular basement membrane include accumulation of Aβ, composite changes, and thickening. The physical properties of the vascular basement membrane carry the potential of obstructing drug delivery to the brain, e.g. thickening of the basement membrane can affect drug delivery to the brain, especially the delivery of nanoparticles.

Neuroprotection strategies to improve stroke outcome have been successful in the laboratory but not in clinical stroke trials, and thus have come under scrutiny by the medical community. Experimental stroke investigators are therefore under increased pressure to resolve this problem. Acute ischemic stroke represents a severe form of metabolic stress that activates many pathological processes and thereby impairs cellular functions. Traditionally, neuroprotection strategies were designed to improve stroke outcome by interfering with pathological processes triggered by ischemia. However, stroke outcome is also dependent on the brain’s capacity to restore cellular functions impaired by ischemia, and this capacity declines with age. It is, therefore, conceivable that this age-dependent decline in the brain’s self-healing capacity contributes to the disparity between the success of neuroprotective strategies in young animals, and limited success in elderly stroke patients. Here, prosurvival pathways that restore protein homeostasis impaired by ischemic stress should be considered, because their capacity decreases with increasing age, and maintenance of proteome fidelity is pivotal for cell survival. Boosting such prosurvival pathways pharmacologically to restore protein homeostasis and, thereby, cellular functions impaired by ischemic stress is expected to counterbalance the compromised self-healing capacity of aged brains and thereby help to improve stroke outcome.

Diffusion weighted imaging (DWI) has been commonly used in acute stroke examination, yet a portion of DWI lesion may be salvageable. Recently, it has been shown that diffusion kurtosis imaging (DKI) defines the most severely damaged DWI lesion that does not renormalize following early reperfusion. We postulated that the diffusion and kurtosis lesion mismatch experience heterogeneous hemodynamic and/or metabolic injury. We investigated tissue perfusion, pH, diffusion, kurtosis and relaxation from regions of the contralateral normal area, diffusion lesion, kurtosis lesion and their mismatch in an animal model of acute stroke. Our study revealed significant kurtosis and diffusion lesion volume mismatch (19.7 ± 10.7%, P < 0.01). Although there was no significant difference in perfusion and diffusion between the kurtosis lesion and kurtosis/diffusion lesion mismatch, we showed lower pH in the kurtosis lesion (pH = 6.64 ± 0.12) from that of the kurtosis/diffusion lesion mismatch (6.84 ± 0.11, P < 0.05). Moreover, pH in the kurtosis lesion and kurtosis/diffusion mismatch agreed well with literature values for regions of ischemic core and penumbra, respectively. Our work documented initial evidence that DKI may reveal the heterogeneous metabolic derangement within the commonly used DWI lesion.

Positron emission tomography with ¹⁵O-labeled gases (¹⁵O-PET) is important for in vivo measurement of cerebral oxygen metabolism both in clinical and basic settings. However, there are currently no reports concerning ¹⁵O-PET in mice. Here, we developed an ¹⁵O-PET method applicable to mice with spontaneous respiration of ¹⁵O-gas without a tracheotomy catheter. Sequential ¹⁵O-PET was also performed in a mouse model of chronic cerebral hypoperfusion with bilateral common carotid artery stenosis (BCAS) induced by placement of microcoils. ¹⁵O-gas with isoflurane was supplied to the nose of mouse with evacuation of excess ¹⁵O-gas surrounding the body. ¹⁵O-PET was performed on days 3, 7, 14, 21, and 28 after surgery. Cerebral blood flow (CBF), cerebral blood volume, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO₂) were calculated in whole brains. A significant decrease in CBF and compensatory increase in OEF in the BCAS group produced CMRO₂ values comparable to that of the sham group at three days post-operation. Although CBF and OEF in the BCAS group gradually recovered over the first 28 days, the CMRO₂ showed a gradual decrease to 68% of sham values at 28 days post-operation. In conclusion, we successfully developed a noninvasive ¹⁵O-PET method for mice.

Cerebral collaterals are dynamically recruited after arterial occlusion and highly affect tissue outcome in acute ischemic stroke. We investigated the efficacy and safety of four pathophysiologically distinct strategies for acute modulation of collateral flow (collateral therapeutics) in the rat stroke model of transient middle cerebral artery (MCA) occlusion. A composed randomization design was used to assign rats (n = 118) to receive phenylephrine (induced hypertension), polygeline (intravascular volume load), acetazolamide (cerebral arteriolar vasodilation), head down tilt (HDT) 15° (cerebral blood flow diversion), or no treatment, starting 30 min after MCA occlusion. Compared to untreated animals, treatment with collateral therapeutics was associated with lower infarct volumes (62% relative mean difference; 51.57 mm³ absolute mean difference; p < 0.001) and higher chance of good functional outcome (OR 4.58, p < 0.001). Collateral therapeutics acutely increased cerebral perfusion in the medial (+40.8%; p < 0.001) and lateral (+19.2%; p = 0.016) MCA territory compared to pretreatment during MCA occlusion. Safety indicators were treatment-related mortality and cardiorespiratory effects. The highest efficacy and safety profile was observed for HDT. Our findings suggest that acute modulation of cerebral collaterals is feasible and provides a tissue-saving effect in the hyperacute phase of ischemic stroke prior to recanalization therapy.

The visualization of cerebral microvessels is essential for understanding brain remodeling after stroke. Injection of dyes allows for the evaluation of perfused vessels, but has limitations related either to incomplete microvascular filling or leakage. In conventional histochemistry, the analysis of microvessels is limited to 2D structures, with apparent limitations regarding the interpretation of vascular circuits. Herein, we developed a straight-forward technique to visualize microvessels in the whole ischemic mouse brain, combining the injection of a fluorescent-labeled low viscosity hydrogel conjugate with 3D solvent clearing followed by automated light sheet microscopy. We performed transient middle cerebral artery occlusion in C57Bl/6j mice and acquired detailed 3D vasculature images from whole brains. Subsequent image processing, rendering and fitting of blood vessels to a filament model was employed to calculate vessel length density, resulting in 0.922 ± 0.176 m/mm³ in healthy tissue and 0.329 ± 0.131 m/mm³ in ischemic tissue. This analysis showed a marked loss of capillaries with a diameter ≤ 10 µm and a more moderate loss of microvessels in the range > 10 and ≤ 20 µm, whereas vessels > 20 µm were unaffected by focal cerebral ischemia. We propose that this protocol is highly suitable for studying microvascular injury and remodeling post-stroke.

A characteristic feature of complex healthy biological systems is the ability to react and adapt to minute changes in the environment. This ‘complexity’ manifests itself in highly irregular patterns of various physiological measurements. Here, we apply Multiscale Entropy (MSE) analysis to assess the complexity of systemic and cerebral near-infrared spectroscopy (NIRS) signals in a cohort of 61 critically ill preterm infants born at median (range) gestational age of 26 (23–31) weeks, before 24 h of life. We further correlate the complexity of these parameters with brain injury and mortality. Lower complexity index (CoI) of oxygenated haemoglobin (HbO₂), deoxygenated haemoglobin (Hb) and tissue oxygenation index (TOI) were observed in those infants who developed intraventricular haemorrhage (IVH) compared to those who did not (P = 0.002, P = 0.010 and P = 0.038, respectively). Mean CoI of HbO₂, Hb and total haemoglobin index (THI) were lower in those infants who died compared to those who survived (P = 0.012, P = 0.004 and P = 0.003, respectively). CoI-HbO₂ was an independent predictor of IVH (P = 0.010). Decreased complexity of brain signals was associated with mortality and brain injury. Measurement of brain signal complexity in preterm infants is feasible and could represent a significant advance in the brain-oriented care.

To assess the true effect of novel therapies for ischaemic stroke, a positive control that can validate the experimental model and design is vital. Hypothermia may be a good candidate for such a positive control, given the convincing body of evidence from animal models of ischaemic stroke. Taking conditions under which substantial efficacy had been seen in a meta-analysis of hypothermia for focal ischaemia in animal models, we undertook three randomised and blinded studies examining the effect of hypothermia induced immediately following the onset of middle cerebral artery occlusion on infarct volume in rats (n = 15, 23, 264). Hypothermia to a depth of 33℃ and maintained for 130 min significantly reduced infarct volume compared to normothermia treatment (by 27–63%) and depended on ischaemic duration (F(3,244) = 21.242, p < 0.05). However, the protective effect varied across experiments with differences in both the size of the infarct observed in normothermic controls and the time to reach target temperature. Our results highlight the need for sample size and power calculations to take into account variations between individual experiments requiring induction of focal ischaemia.

Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood–brain barrier.

Angiogenesis is a critical compensation route, which has been demonstrated in the brain following ischemic stroke; however, few studies have investigated angiogenesis in chronic intracranial atherosclerosis disease (ICAD). We used ⁶⁸Ga-NOTA-PRGD2 positron emission tomography/computed tomography based imaging to detect angiogenesis in chronic ICAD and to explore the factors that may have affected it. A total of 21 participants with unilateral severe chronic ICAD were included in the study. Of the 21 participants, 19 were men; the mean (SD) age was 52 (15) years. In 18 participants, we observed elevated ⁶⁸Ga-NOTA-PRGD2 uptake in the peri-infarct, subcortical, and periventricular regions of the lesioned side, with a higher ⁶⁸Ga-NOTA-PRGD2 SUV_max compared to that in the contralateral hemisphere (0.15 vs. 0.06, p=0.001). The ¹⁸F-FDG PET SUV_max was significantly lower on the lesioned side (11.28 vs. 13.92, p=0.001). Subgroup analyses revealed that the recent group (<6 months) had a higher lesion-to-contralateral region ratio SUV_max than the remote group (>6 months) (6.73 vs. 2.36, p<0.05). Our results provide molecular imaging evidence of angiogenesis in patients with severe chronic ICAD. Furthermore, the extent of angiogenesis in chronic ICAD may be affected by the post-qualified event time interval, and not by infarction itself or the severity of the arterial lesion.

Neurovascular changes are likely to interact importantly with the neurodegenerative process in idiopathic Parkinson’s disease (IPD). Markers of neurovascular status (NVS) include white matter lesion (WML) burden and arterial spin labelling (ASL) measurements of cerebral blood flow (CBF) and arterial arrival time (AAT). We investigated NVS in IPD, including an analysis of IPD clinical phenotypes, by comparison with two control groups, one with a history of clinical cerebrovascular disease (CVD) (control positive, CP) and one without CVD (control negative, CN). Fifty-one patients with IPD (mean age 69.0 ± 7.7 years) (21 tremor dominant (TD), 24 postural instability and gait disorder (PIGD) and six intermediates), 18 CP (mean age 70.1 ± 8.0 years) and 34 CN subjects (mean age 67.4 ± 7.6 years) completed a 3T MRI scan protocol including T₂-weighted fluid-attenuated inversion recovery (FLAIR) and ASL. IPD patients showed diffuse regions of significantly prolonged AAT, small regions of lower CBF and greater WML burden by comparison with CN subjects. TD patients showed lower WML volume by comparison with PIGD patients. These imaging data thus show altered NVS in IPD, with some evidence for IPD phenotype-specific differences.

The purpose of this study was to explore the capability and uniqueness of amide proton transfer-weighted (APTw) imaging in the detection of primary and secondary injury after controlled cortical impact (CCI)-induced traumatic brain injury (TBI) in rats. Eleven adult rats had craniotomy plus CCI surgery under isoflurane anesthesia. Multi-parameter MRI data were acquired at 4.7 T, at eight time points (1, 6 h, and 1, 2, 3, 7, 14, and 28 days after TBI). At one and six hours post-injury, average APTw signal intensities decreased significantly in the impacted and peri-lesional areas due to tissue acidosis. A slightly high APTw signal was seen in the core lesion area with respect to the peri-lesional area, which was due to hemorrhage, as shown by T₂*w. After the initial drop, the APTw signals dramatically increased in some peri-lesional areas at two and three days post-injury, likely due to the secondary inflammatory response. The use of APTw MRI has the potential to introduce a novel molecular neuroimaging approach for the simultaneous detection of ischemia, hemorrhage, and neuroinflammation in TBI.

Cerebral amyloid angiopathy (CAA) is a small-vessel disease preferentially affecting posterior brain regions. Recent evidence has demonstrated the efficacy of functional MRI in detecting CAA-related neurovascular injury, however, it is unknown whether such perturbations are associated with changes in the hemodynamic response function (HRF). Here we estimated HRFs from two different brain regions from block design activation data, in light of recent findings demonstrating how block designs can accurately reflect HRF parameter estimates while maximizing signal detection. Patients with a diagnosis of probable CAA and healthy controls performed motor and visual stimulation tasks. Time-to-peak (TTP), full-width at half-maximum (FWHM), and area under the curve (AUC) of the estimated HRFs were compared between groups and to MRI features associated with CAA including cerebral microbleed (CMB) count. Motor HRFs in CAA patients showed significantly wider FWHM (P = 0.006) and delayed TTP (P = 0.03) compared to controls. In the patient group, visual HRF FWHM was positively associated with CMB count (P = 0.03). These findings indicate that hemodynamic abnormalities in patients with CAA may be reflected in HRFs estimated from block designs across different brain regions. Moreover, visual FWHM may be linked to structural MR indications associated with CAA.

This study proposes a method for territorial segmentation and volumetric flow rate (VFR) distribution measurement of cerebral territories based on time-resolved contrast enhanced magnetic-resonance-angiography (MRA). The method uses an iterative region-growing algorithm based on bolus-arrival-time with increased temporal resolution. Eight territories were segmented: (1) right and (2) left internal carotid arteries, including the middle cerebral artery (ICA+MCA), excluding the anterior cerebral arteries (ACA); (3) right and left ACA (R+L-ACA); (4) right and (5) left external carotid arteries (ECA); (6) right and (7) left posterior cerebral arteries (PCA); and (8) vertebrobasilar territory. VFR percentage, relative to the entire brain (rVFR), was measured based on territorial volume as a function of time. Mean rVFR values of fifteen healthy subjects were: ICA+MCA = 23 ± 2%, R + L-ACA = 17 ± 3%, ECA = 4 ± 2%, PCA = 12 ± 2%, and vertebrobasilar territory = 31 ± 4%. Excluding the ECA-rVFR, which is underestimated, these values are comparable to previously reported values. Six subjects were scanned twice, demonstrating comparable and even higher reproducibility than previously reported using phase-contrast, yet with faster scan time (∼1 min). This method was implemented in one patient with MCA occlusion and one with Moyamoya syndrome scanned before and after bypass surgery, demonstrating its clinical potential for quantitative assessment of the degree of occlusion and the effect of surgery.